Complexation of fatty acid-conjugated molecules with albumin

ABSTRACT

The technology described herein is directed to conjugating various therapeutic or functional molecules, such small chemical drugs, peptides, oligonucleotides, isotopes and imaging regents with fatty acids, such as LCFAs. The fatty acid-conjugated molecules are complexed with a human serum albumin protein in vitro.

RELATED APPLICATIONS

This application is a 371 National Stage of International ApplicationNo. PCT/US2008/012943, filed Nov. 20, 2008, which claims priority toU.S. Provisional App. No. 61/004,056, filed Nov. 21, 2007. Theaforementioned patent applications are expressly incorporated herein byreference in its entirety.

BACKGROUND

The invention relates generally to the field of delivery of drugs,vectors, and other molecules in a variety of settings, includingtherapeutic, diagnostic, research and clinical uses.

Small organic molecules (i.e., those having molecular weights <10,000,and frequently <5,000 or <1,000) have well known uses as drugs in avariety of human ailments. Peptide and oligonucleotide drugs (including,for example, antisense oligonucleotides, ribozymes, aptamers, andsiRNA/shRNA molecules) have emerged as powerful tools for drug targetvalidation and promising therapeutics for a wide variety of humandiseases. However, realization of the promise these agents offer asdrugs has been hindered by the lack of efficient methods of deliveringthem to physiological sites at which they may exert useful activity.Obstacles to efficient delivery include considerations of in vivostability, tissue and cell specificity, intracellular durability,immunogenicity, and toxicity of the agents. Many well-characterizedsmall chemical drugs confront the same delivery bottlenecks that limittheir potency in human applications.

Similarly, inorganic drugs (e.g., radioisotopes andradioisotope-containing compounds), imaging agents, and other smallmolecules intended for delivery to cells exhibit many of the samedrawbacks with regard to their stability and delivery to desired cellsand tissues.

Human Serum Albumin

Human serum albumin (HSA) is the most abundant protein in human plasma.HSA is known to bind an extraordinarily wide range of metabolites anddrugs. Binding between HSA and such compounds affects, sometimesdramatically, their pharmacokinetics and pharmacodynamics.

HSA is synthesized in the liver and secreted as a non-glycosylatedprotein. It accounts for 60% of the mass of the plasma proteins and ispresent in the blood at a concentration around 0.6 mM with an averagehalf-life of 14 days. Although the critical function of HSA inmaintaining normal colloid osmotic pressure in plasma and ininterstitial fluid is recognized, the molecular mechanisms of its basicphysiological functions, such as metabolite transportation, exogenouschemical binding, and antioxidant protection, are not fully understood.

HSA is synthesized as a 585-residue single chain globular proteinlacking prosthetic groups and glycosylation. The primary amino acidsequence of HSA is shown in FIG. 1. HSA has three homologous domains(designated domains I, II and III, as indicated in FIG. 2) that foldinto the shape of a heart and each domain is classified into twosubdomains (A and B). Further details of the structure of domains I, II,and III are shown in FIG. 3. Alpha-helixes account for approximately 67%of the secondary structure of HSA, and no beta-sheet secondary structureoccurs in HSA. A unique feature of HSA is its 35 cysteine residues, 34of which form 17 disulfide bonds. The only free cysteine (Cys-34, i.e.,the cysteine residue that occurs at residue 34 of HSA) contributessignificantly to the antioxidant activity of HSA and this residue can bechemical modified in a variety of known ways. The disulfide bonds arealso responsible for the thermal stability of HSA. Others haverecognized that the primary polypeptide structure of HSA includesseveral loops that are tightly held by disulfide bonds and present onthe external surface of the protein.

Almost every body fluid contains some amount of HSA. In addition, HSAoccurs within cells like ovarian cells, brain cells, peripheral nervecells, lymphocytes, macrophages, and other cells. Tumor cells often takeup HSA to a greater extent than non-tumorous cells of the same type. Forexample, albumin makes up 19% of the soluble protein of breast cancercells.

Due to its availability, biocompatibility, nontoxicity, andimmunogenicity, human serum albumin (HAS) has been used as a stabilizerin biopharmaceutical products (vaccine and recombinant protein), anadjuvant in drug formulation and a component of imaging agents. It alsocan be used to coat biomaterial surface and purify chiral chemicals.Moreover, adding signal peptides or functional compounds to albumin bychemical modification has been commonly used for drug targeting anddelivery research. Using chemical crosslinking, HSA can also beformulated into microspheres and nanoparticles to encapsulate drugs,oligonucleotides, and radioisotopes for delivery or diagnosis purpose.However problems associated with the chemical modification may severelyhinder the clinical application of chemically modified albumins. First,the modification is non-specific and non-homogenous. Second, thesemodifications change the physicochemical and biochemical properties ofalbumins and may result in an immune response. Third, the chemicallymodified albumin may not fold properly and exhibit an abnormal surfacecharge distribution. These changes are likely to be recognized byendogenous albumin and other serum proteins and cause aggregation andrapid elimination in vivo.

Long Chain Fatty Acids

Long chain fatty acids (LCFAs, i.e., carboxylic acids having annon-branched aliphatic chain having 16-20 carbon atoms in its backbone)are essential for many cellular functions. LCFAs serve as an importantenergy resource and are also critical components of lipids, hormones,and proteins. LCFAs are known to be bound and transported by HSAs withinthe human body.

Formation of conjugates between LCFAs and many small molecules is knownto enhance the serum stability and delivery of the small molecules by amechanism facilitated by binding of the small molecule-LCFA conjugatewith HSA.

Shortcomings in stability, solubility, and ‘targetability’ limit theutility of many potentially useful drugs, diagnostic agents, nucleicacid vectors, and other relatively small molecules within the humanbody. The technology disclosed herein overcomes these shortcomings.

BRIEF SUMMARY OF THE INVENTION

The disclosure relates to recombinant animal albumin proteins, such ashuman serum albumin (HSA). The recombinant proteins are fusion proteinshaving a ligand-binding protein domain inserted in place of a surfaceloop of the native (i.e., naturally-occurring form of the) protein. Theligand bound by the domain can be any of a wide variety of ligandsincluding, for example, ligands that occur on the surface of a cell ofan animal, ligands that occur in a tissue of an animal. Examples of suchdomains include RGD-containing domains, adrenomedulin domains,endothelin-1 domains, matrix metalloproteinase 9 binding peptidedomains, matrix metalloproteinase 2 binding peptide domains, andaminopeptidase N binding peptide domains.

The surface domains of animal albumin proteins are known. For examplethe surface loops of HSA have the amino acid sequences disclosed hereinas SEQ ID NOs: 3-11. With reference to the naturally-occurring aminoacid sequence of HSA (e.g., as shown in FIG. 1), the surface loops ofHSA include (i) the loop defined by residues 53-62; (ii) the loopdefined by residues 75-91; (iii) the loop defined by residues 91-101;(iv) the loop defined by residues 168-177; (v) the loop defined byresidues 245-253; (vi) the loop defined by residues 265-279; (vii) theloop defined by residues 278-289; (viii) the loop defined by residues360-369; (ix) the loop defined by residues 437-448; (x) the loop definedby residues 461-477; (xi) the loop defined by residues 476-487; and(xii) the loop defined by residues 558-567 of SEQ ID NO: 1.

The recombinant albumins described herein can be in the form of acomplex that includes the albumin complexed with a compound that bindsnon-covalently therewith. The compound can be a conjugate of firstmolecule and a fatty acid. Examples of suitable first molecules includepolynucleotides, polypeptides, and drugs other than polynucleotides orpolypeptides. Other suitable examples of first molecules includeradiolabeled compounds and imaging agents other than radiolabeledcompounds. The fatty acid can be a C₁₀-C₂₀ fatty acid or, preferably, aC₁₆-C₂₀ fatty acid.

Another aspect of this disclosure pertains to a method of targeting afirst molecule to a ligand. This method involves contacting a firstcomposition and a second composition. The first composition includes aconjugate of the first molecule and a fatty acid. The second compositionincludes a recombinant animal albumin, as described herein. The firstand second compositions are contacted for a time and under conditionssufficient for the conjugate to bind with the albumin to form a complex.After contacting the first and second compositions, the complex and theligand are contacted. The ligand-binding domain of the albumin bindswith the ligand and targets the first molecule to the ligand.

Another aspect of the technology described herein relates to a method ofdelivering a first molecule to a target within the body of a human. Thismethod involves contacting a first composition and a second compositionoutside the body of the human. The first composition includes aconjugate of the first molecule and a fatty acid. The second compositionincludes a human serum albumin (i.e., naturally occurring HSA or arecombinant HSA of the type described herein). The first and secondcompositions are contacted for a time and under conditions sufficientfor the conjugate to bind with the albumin to form a complex.Thereafter, the complex is administered to a tissue from which thetarget is accessible, and the first molecule is thereby delivered to thetarget. Suitable targets include cells and tissues of the human, andviruses, bacteria, parasites within the human. A preparation thatincludes the complex can be applied directly to a tissue wherein thetarget is located (or directly to a tissue that is itself the target).Alternatively, the preparation can be applied to an endothelial tissuethat separates the target from the site of application. As anotheralternative, the preparation can be administered systemically (e.g.,intravenously).

The albumin used in these methods can be normal human serum albumin,which can optionally be substantially purified, defatted, or both, priorto use in the methods. In one embodiment, substantially all firstmolecules not bound with the albumin after contacting the first andsecond compositions are removed from the preparation containing thecomplex prior to administering the preparation to the target.Furthermore, more than one conjugate can be bound with the albumin.

In another aspect, this disclosure relates to a method of enhancingdelivery of a first molecule to a cell of a human prior to administeringthe first molecule to the human. The method involves conjugating thefirst molecule and a fatty acid to form a conjugate. A first compositionthat includes the conjugate and a second composition are contactedoutside the body of the human. The second composition comprises a humanserum albumin (native HSA or a recombinant HSA as described herein). Thefirst and second compositions are contacted for a time and underconditions sufficient for the conjugate to bind with the albumin to forma complex. In the form of the complex, the first molecule exhibitsenhanced delivery to human cells.

This disclosure further relates to compositions for delivering a firstmolecule to a cell of a human. The composition includes a human serumalbumin (a native HSA or a recombinant HSA of the type described herein)having bound thereto a conjugate. The conjugate includes a fatty acidmoiety conjugated with the first molecule. In this form, the firstmolecule can be delivered to a cell of a human. Preferably, thecomposition contains substantially no first molecules not bound with thealbumin. In one embodiment, the albumin also has bound thereto a secondconjugate. The second conjugate includes a second fatty acid moietyconjugated with a compound that binds specifically with a targetmolecule that occurs on the surface of the cell. In another embodiment,the second conjugate includes a second fatty acid moiety conjugated witha compound that binds specifically with a target molecule that occurs ina tissue that includes the cell.

BRIEF SUMMARY OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a listing of the primary amino acid sequence (SEQ ID NO: 1) ofhuman serum albumin (HSA). Residues 1 to 18 of SEQ ID NO: 1 are a signalpeptide sequence, and residues 19 to 24 are a portion of the HSApropeptide that is cleaved from the mature protein, which has the aminoacid sequence of residues 25 to 609 of SEQ ID NO: 1.

FIG. 2 is a diagram depicting the domain structure of HSA andreplacement of a surface loop of HSA with an exogenous peptide sequence.In the diagram, the three lobular domains I, II, and III of HSA aredepicted as aggregations of three sub-domains (open ovals). Peptideloops (open arcs) of HSA connect the sub-domains to one another and arelocated at or near the external (i.e., solvent-exposed) surface of theprotein. In the diagram, an exogenous peptide domain (hatched arc) issubstituted in place of one of the surface loops in domain I. Theexogenous peptide domain has a ligand-binding domain that bindsspecifically with a particular ligand, shown in this diagram as aprotein (P).

FIG. 3, consisting of FIGS. 3A, 3B, 3C, 3D, and 3E depicts HSA protein(FIGS. 3A-3C) and a recombinant HSA (rHSA; FIGS. 3D and 3E) protein intowhich an exogenous RGD-motif-containing polypeptide sequence has beeninserted in place of a surface loop of HSA. FIGS. 3A, 3B, and 3B,illustrate the structures of domains I, II, and III (respectively) ofnaturally-occurring HSA. FIG. 3A shows the sequence (SEQ ID NO: 2) andloop structure of residues 53-62 of HSA. The disulfide linkage betweenthe side chains of cysteine residues 53 and 62 is shown as a solid bar,and residues 1-52 and 62+ are not all individually listed. Shown in FIG.3D are the sequences of RDC-4C (a RGD motif held by two pairs ofdisulfide bonds; SEQ ID NO: 3) of and an RGD-motif-containing portion(SEQ ID NO: 4) of fibronectin, either of which can be inserted in placeof residues 53-62 of HSA. FIG. 3E is a ribbon model of thethree-dimensional structure of the rHSA, in which disulfide bonds in theHSA structure are shown in red and residues 53-62 of HSA are shown indark blue. The figure also shows the sequence (SEQ ID NO: 2) and loopstructure of residues 53-62 of HSA and the RGD-4C sequence (SEQ ID NO:3) and RGD-motif-containing portion of fibronectin (SEQ ID NO: 4).

FIG. 4 is a diagram that depicts a cloning strategy described herein.

FIG. 5 is a diagram that depicts primers for use in a cloning strategydescribed herein.

FIG. 6 consists of FIGS. 6A and 6B. FIG. 6A shows the sequence (SEQ IDNO: 12) of the FRI5′ primer. FIG. 6B shows the sequence (SEQ ID NO: 13)of a variant of this primer.

FIG. 7 shows the sequences (SEQ ID NO: 14 in FIG. 7A; SEQ ID NO: 15: inFIG. 7B, and SEQ ID NO: 16 in FIG. 7C) of FRII3′ primers describedherein.

FIG. 8 shows the sequences (SEQ ID NO: 17 in FIG. 8A; SEQ ID NO: 18 inFIG. 8B, and SEQ ID NO: 19 in FIG. 8C) of FRI3′ and FRII 5′ primersdescribed herein.

FIG. 9 shows the sequences (SEQ ID NO: 22 in FIG. 9A; SEQ ID NO: 23 inFIG. 9B, and SEQ ID NO: 24 in FIG. 9C) of FRI3′ and FRII 5′ primersdescribed herein.

FIG. 10 shows the sequences (SEQ ID NO: 25 in FIG. 10A; SEQ ID NO: 26 inFIG. 10B, and SEQ ID NO: 27 in FIG. 10C) of FRI3′ and FRII 5′ primersdescribed herein.

FIG. 11 shows the sequences (SEQ ID NO: 34 in FIG. 11A; SEQ ID NO: 35 inFIG. 11B, and SEQ ID NO: 36 in FIG. 11C) of FRI3′ and FRII 5′ primersdescribed herein.

FIG. 12 shows the sequences (SEQ ID NO: 37 in FIG. 12A; SEQ ID NO: 38 inFIG. 12B, and SEQ ID NO: 39 in FIG. 12C) of FRI3′ and FRII 5′ primersdescribed herein.

FIG. 13 shows the sequences (SEQ ID NO: 40 in FIG. 13A; SEQ ID NO: 41 inFIG. 13B, and SEQ ID NO: 42 in FIG. 13C) of FRI3′ and FRII 5′ primersdescribed herein.

FIG. 14 shows the sequences (SEQ ID NO: 43 in FIG. 14A; SEQ ID NO: 44 inFIG. 14B, and SEQ ID NO: 45 in FIG. 14C) of FRI3′ and FRII 5′ primersdescribed herein.

FIG. 15 shows the sequences (SEQ ID NO: 46 in FIG. 15A; SEQ ID NO: 47 inFIG. 15B, and SEQ ID NO: 48 in FIG. 15C) of FRI3′ and FRII 5′ primersdescribed herein.

FIG. 16 shows the sequences (SEQ ID NO: 49 in FIG. 16A; SEQ ID NO: 50 inFIG. 16B, and SEQ ID NO: 51 in FIG. 16C) of FRI3′ and FRII 5′ primersdescribed herein.

FIG. 17 shows the sequences (SEQ ID NO: 54 in FIG. 17A; SEQ ID NO: 55 inFIG. 17B) of an FRI3′ primer and an HSA II fragment described herein.

FIG. 18 is a bar graph that shows the results of esterase assaysdescribed herein.

FIG. 19 shows the results of an αvβ3 (alpha v beta 3) integrin-bindingassay described herein.

DETAILED DESCRIPTION

The technology disclosed herein relates to compositions and methods inwhich a fatty acid-conjugated compound is complexed with a human serumalbumin (HSA) protein.

The complexes can be put to a wide variety of uses, depending on thecomponents from which the complex is formed. For example, the complexesdescribed herein are useful for improving delivery of drugs, imagingagents, and other molecules to cells of a human. The complexes are alsouseful as reagents for detection of various targets in biological andlaboratory systems. Because moieties that exhibit highly specificbinding affinities can be incorporated into the HSA, the complexes canbe used as an alternative to antibodies for a wide variety of purposesfor which antibodies are used. Albumin proteins modified to have such amoiety incorporated in place of a surface loop thereof are referred toherein as “target-binding albumins.” Target-binding albumins can be usedalone, or complexed with a fatty acid or a fatty acid-conjugatedcompound as a substitute for an antibody that exhibits a similartarget-binding specificity.

The technology makes use of the native biochemical and physiologicalcharacteristic of human serum albumin (HSA) and fatty acids. Theprocesses of conjugating molecules with fatty acids and of formulatingHSAs having fatty acid-conjugated molecules bound thereto is simple, andthe resulting compositions are simple and easy to manufacture. Thisdisclosure is made in the context of HSA, which is suitable for in vivouse in humans. The technology can also be employed in substantially thesame way to make complexes using fatty acid-conjugated albumins of otheranimals for in vivo use in those animals. For in vitro uses (i.e., inwhich introduction of the complexes into the body of an animal is notcontemplated), the identity of the albumin used is notcritical—substantially any albumin, as modified herein, can be used insuch circumstances.

Advantage can be taken of ability of HSA to bind and transport nativelong chain fatty acids (LCFAs) and, significantly, molecules conjugatedwith LCFAs. Functional and therapeutic molecules can be delivered tobody tissues in the form of fatty acid-conjugates, the conjugate beingbound in a complex with HSA or in complex with a recombinant HSA (rHSA),as described herein. This approach differs from simply using fattyacid-conjugated molecules, which has been described by others. Fattyacid-conjugated polypeptides have been reported, for example.

As described herein, instead of directly administrating the conjugate toa human or other subject in vivo, the conjugate is first formulated withHSA in vitro under conditions that can be selected to favor (oroptimize) formation of a complex between the conjugate and the HSA. Thecomplex is thereafter delivered to a subject in vivo, eithersystemically or locally, or used in an in vitro application (e.g., invitro delivery of a polypeptide or oligonucleotide to a cell).Non-complexed conjugate can be reduced or eliminated (e.g., bypurification such as chromatography) in the preparation prior to itsuse. Complexation reduces or substantially eliminates binding of theconjugate with serum proteins of the subject in in vivo applications,and can reduce or eliminate undesirable binding of the conjugate withother components of an in vitro system. The complexation also reduces oreliminates enzyme degradation, renal excretion, and other undesirablefates that might befall the conjugate were it administered in anon-complexed form. The methods can also be used to reduce or preventdelivery of a conjugate to tissues in which the conjugate (or thede-conjugated molecule) has detrimental effects.

In in vivo applications, the conjugate-HSA complex described herein canbe transported across endothelial cells by the process of transcytosis.The complexes made by the processes described herein can thereby reachthe interstitial space of tissues in vivo, which represents animprovement in the ability of practitioners to target therapeutic andprophylactic agents to these body compartments. This property enablesthe complex to be administered to a patient by a wide variety of routesand in a wide variety of dosage forms. The complexes can be used totarget a wide variety of tissues, such as solid tissues and blood cells,as well as other targets such as bacteria, viruses, and non-livingmaterials. The complexes can be constructed, as described herein, tospecifically bind with any of a wide variety of proteins, metabolites,or other molecules.

There are at least several benefits of the technology described herein.Probably chief among the benefits are the facts that HSA isbiocompatible and its immunogenicity is limited. HSA-complexed, fattyacid-conjugated molecules share that biocompatibility and limitedimmunogenicity. On account of these properties, the methods describedherein can be used to improve delivery and half-life and to reducetoxicity and side effects of pharmaceutical compounds (i.e., moleculeswhich exhibit one or more therapeutic, diagnostic, and prophylacticeffects on animal bodies) administered to animals. Such benefits can beachieved by conjugating the molecule with a fatty acid (preferably aC₁₆-C₂₀ LCFA) and complexing the resulting conjugate with an HSA(including an rHSA having an exogenous portion bearing a ligand-bindingdomain) prior to administering the compound to a subject. SuchHSA-complexed, fatty acid-conjugated molecules can, instead of targetingdelivery of a compound to a discrete body tissue, be used in vivo ascomplexes that bind particular cells or molecules that are believed orexpected to be present in a biological system.

The technology described herein is a platform technology, and has abroad range of applications in drug formulation and delivery,radiotherapy, antibody-like targeting, proteomic analysis, biosensing,bioimaging, biomaterial processing, and other fields.

In vitro formulation of a complex of an HSA and a fatty acid-conjugatedmolecule has a number of advantageous features. For example, suchformulation can greatly enhance serum solubility of the molecule,especially for lipophilic molecules. Such formulations also reducerandom binding of the molecule (or the conjugate) with other serumproteins in vivo, and reduce elimination of the molecule (or conjugate)occasioned by binding with other serum proteins. Formulating a moleculein this way can prolong the time that the molecule remains in thecirculation. Molecules formulated in this way can also be delivered morespecifically to tissues or types of tissues than molecules not soformulated.

By way of example, HSA-complexed, fatty acid-conjugated molecules can betransported to tissues separated from the circulation by an interposedendothelial membrane by way of transendothelial transcytosis.

Further by way of example, fatty acid-conjugated molecules complexedwith a rHSA which has an exogenous portion which binds specifically witha tissue target (e.g., a cell-surface protein) can be deliveredspecifically to cells or tissues that bear the target and that areaccessible to HSA.

Still further by way of example, target-binding albumins describedherein can be used to encapsulate drugs or other materials in order totake advantage of the binding specificity of the target-binding albumin.Such compositions can optionally include a fatty acid-conjugatedmolecule (e.g., the encapsulated material or another compound) complexedwith the target-binding albumin.

Because many cell types are able to take up HSA, the HSA-complexed,fatty acid-conjugated molecules described herein can be used to deliverthe molecules to such cell types, even without adding an exogenoustargeting domain to the HSA. This disclosure describes compositions andmethods for formulating pharmaceutically active molecules (and otherbiocompatible molecules, such as imaging agents) that are versatile, inthat any of a wide variety of molecules can be conjugated with any of abroad range of fatty acids, and in that the resulting conjugates can becomplexed with naturally-occurring, synthetic, or recombinant HSAproteins. The rHSAs can include any of a wide variety of ligand-bindingdomains within their structure. The ligands that can be bound with suchdomains include substantially all of those that can be bound by aprotein sequence insertable within the HSA surface domains describedherein, and include small molecules, proteins, cells, extracellularmatrix materials, metal ions, and others.

In addition to their uses as compositions to be administered directly tosubjects, the composition described herein can be used to coat one ormore surfaces of a biomaterial. Such coating improves thebiocompatibility of the object (e.g., a drug nano- or micro-particle) ofwhich the surface is a part. Such coatings can also be used to attractcertain molecules or cells to the surface or to repel them from thesurface. The coatings can be made by binding a fatty acid or fattyacid-conjugated molecule to a surface, and thereafter contacting the HSA(or rHSA) therewith. The coatings can also be made by binding the HSA(or rHSA) with the surface, and thereafter contacting the surface with afatty acid or fatty acid-conjugated molecule. Similarly, the surface canbe coated with a HSA (or rHSA) having a fatty acid or fattyacid-conjugated molecule already complexed therewith. If the rHSA is atarget-binding albumin, then the material coated with the target-bindingalbumin can be used in a wide-variety of ways in place of materialscoated with an antibody that binds the same target. Examples of suchmaterials include those used for affinity separation and purificationprocedures, in vitro diagnostic tests, biosensing applications, andbinding of a chromophore, a fluorophore, or another detectable markerwith a target. In this last example, use of the target-binding albuminsdescribed herein can be preferable to use of a comparable antibody, inthat the detectable marker need not be (although it can be) linkeddirectly to the protein (i.e., the antibody or albumin), but can insteadbe conjugated with a fatty acid (or with a fatty-acid conjugatedmolecule) and complexed with the albumin, prior to, during, or aftercontacting the target and the albumin.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

“Human serum albumin” (HSA) refers to the protein generally known by thesame name, having the structure described, for example, in Carter etal., (1989, Science 244(4909):1195-1198, as described in entry no.NP_(—)000468 of the NCBI's GenPept protein sequence database, and havingthe primary amino acid sequence (i.e., residues 25 to 609 of SEQ IDNO: 1) shown in FIG. 1.

“Peptide display” refers to a variety of known technologies wherebybinding between a polypeptide expressed (i.e., “displayed”) in anappropriate vector and a desired target can be screened and wherebyinformation sufficient to reproduce the displayed polypeptide (e.g., agenetic sequence encoding the amino acid sequence of the polypeptide oran identifier correlatable with such a sequence) can be retrieved uponobservation of binding (or non-binding). Peptide display technology iswell known and includes, for example, phage display libraries wherein amultiplicity of virions each express on their surface a polypeptide andwherein binding between the expressed polypeptide and a target can beused to isolate and reproduce the virion expressing the polypeptide(whereby genetic material encoding the polypeptide can be obtained.) Inthe context of this disclosure, phage display technology for analyzingpolypeptides displayed between disulfide-linked cysteine residues is ofsignificant use.

A “recombinant” protein is a protein which has been engineered to have aprimary amino acid sequence that differs from the naturally-occurringprimary amino acid sequence of the protein. For example, a recombinantHSA (rHSA) has a sequence that differs from SEQ ID NO: 1, such as anrHSA having a fibronectin RGD-containing domain inserted in place of asurface loop of HSA, as described herein.

An “exogenous” portion of an amino acid sequence of a protein is aportion of the amino acid sequence that differs from the correspondingportion of the naturally-occurring sequence of the protein as a resultof a change made or induced by man in the nucleic acid sequence thatencodes the protein.

A polypeptide is inserted “in place of” a surface loop of an HSA if atleast most (i.e., >50%, and preferably all) of the contiguous residuesthat normally occur between the disulfide-bound cysteine residues thatdefine the surface loop are absent and replaced by the polypeptide.

Detailed Description

Uptake of long chain fatty acids (LCFAs) is an essential cellularmetabolic process. It is known that cell are able to take up LCFAs bymeans of receptor-mediated binding and transmembrane transportation.LCFAs are also known to taken up by cells by means of passive diffusion.It is also known that cells can translocate LCFA across the cellularmembrane by means of pathways mediated by albumin proteins that arecapable of binding with LCFAs and translocating across cellularmembranes.

The technique of fatty acid conjugation of peptides and other smallmolecules has been used by others to modify therapeutic peptides inorder to prolong in vivo circulation. It is thought that the conjugateis taken up by substantially the same means as LCFAs and thatprolongation of conjugate circulation may be related to complexation ofthe conjugate with albumins present in blood. However, in vitroconjugation of a molecule with a fatty acid, together with (still invitro) complexation of that conjugate with a human serum albumin (HSA)protein has not been previously reported. This in vitroconjugation-and-complexation process is described herein.

The process described herein is simple to perform and can be used for awide variety of molecules to be delivered to a subject. The process isperformed in vitro, and significant control can therefore be exertedover the process, which control cannot be exerted in systems in whichcomplexation of HSA and fatty acid moieties is left to the vagaries ofbiological systems such as the human blood stream and peritoneal cavity.This controlled, in vitro processing permits improved control of thepharmacodynamic and pharmacokinetic profiles of molecules (e.g., drugs)that are included in the complex.

Further described herein are methods of modifying the structure of HSAin order to cause it to bind specifically with a protein or other ligandthat occurs within a subject's body. Molecules formulated with suchrecombinant HSAs (rHSAs) permit targeting of compounds to particularbody locations, to particular types of cells, or to other structuresthat act as a ligands with a protein ligand-binding domain or motif Thetechnology described herein can serve as a platform technology forapplications in drug formulation and delivery, radiotherapy, bioimaging,and biomaterial processing.

The technology described herein is directed to conjugating varioustherapeutic or functional molecules, such small chemical drugs,peptides, oligonucleotides, isotopes and imaging regents with fattyacids, such as LCFAs. The fatty acid-conjugated molecules are then boundto an HSA to form a complex in vitro. This approach differs fromprevious methods, wherein fatty acid-conjugated molecules were used toattempt to achieve improved delivery of the molecules. An aspect of thisprocess, the criticality of which was not previously appreciated, isthat formation of a complex between HSA and the LCFA-molecule complexshould be completed ex vivo—i.e., outside the body of the human to whichthe complex is to be administered. For in vitro uses of the complex, thecomplex is also formed in vitro—i.e., outside the body of any animalfrom which the albumin may be obtained.

Described herein are methods of delivering a molecule to a cell withinthe body of a subject such as a human. The method includes the steps ofcontacting a first composition and a second composition outside the bodyof the subject. The first composition includes a conjugate of themolecule and a fatty acid. The second composition includes a human serumalbumin protein. The two compositions are contacted for a time and underconditions sufficient for the conjugate to bind with the albumin to forma complex.

Compositions made in this way can be administered to a tissue, fluid, orbody compartment from which is accessible the cell to which delivery ofthe molecule is desired. The fact that HSA is found in many human celltypes indicates that the methods and compositions described herein canbe used to deliver molecules to at least those cell types.

The HSA protein used in the compositions and methods described hereincan be engineered to form rHSAs that include within their structure afunctional polypeptide sequence, such as a polypeptide domain that bindsspecifically with a particular ligand or type of ligand. Such functionalpolypeptides can be incorporated in place of one of the surface loopportions of the naturally occurring HSA sequence, the surface loopsbeing defined by the disulfide-linked cysteine residues of HSA that areidentified elsewhere herein. It is immaterial whether thedisulfide-linked cysteine residues that define the boundary of thereplaced surface loop in the rHSAs described herein are derived from anunmodified HSA, derived from the functional polypeptide, or derived fromboth. The disulfide-linkage serves to isolate the secondary- andtertiary-structure-inducing effects of the functional polypeptide fromthe remainder of the rHSA, preserving both the functions andconformation of HSA and the function and conformation of the functionalpolypeptide.

The identity of the ligand and can be selected by the person engineeringthe rHSA. By way of example, suitable ligands include cellular membraneproteins, extracellular matrix proteins, protein markers associated withthe cells, serum proteins and metabolites, body fluid proteins andcomponents, virus proteins, and bacterial proteins.

More than one functional polypeptide sequence can be inserted within anrHSA. For example, an rHSA can have a polypeptide domain that bindsspecifically with a first ligand inserted in place of one of the surfaceloops of the corresponding HSA, a second polypeptide domain that bindswith a second ligand in place of another of the surface loops of theHSA, and so on. More than one polypeptide domain that binds with thesame ligand can be inserted within different surface loops of the HSA,even if the domains bind with the same portion of the ligand such thatbinding of one domain with a ligand molecule precludes or obscuresbinding of the second domain with the same ligand molecule. Clearly,domains that bind with different domains of a single ligand molecule canbe used. Similarly, domains that bind specifically with entirelydifferent ligand molecules can be used.

In the context of a rHSA protein engineered to include a polypeptidesequence that binds with a portion of target protein, it is immaterialwhether the target protein or the corresponding target protein-bindingsequence of the rHSA is referred to as the “ligand.” The target proteincan, for example, be a cell surface receptor protein that specificallybinds with (i.e., has as a “ligand” of the receptor protein) a certainprotein structure, and that structure can be incorporated into the rHSAsurface loop domain. What is important is that the “ligand-bindingdomain” of the rHSA bind specifically with a structure present in thetarget (e.g., a structure present on a cell surface protein of a targetcell type in a human's body). The structure to which that domain bindscan be a protein, or it can be any other molecules known to bindspecifically with protein domains, such as carbohydrate structures,extracellular matrix components, mineral substrates (e.g., bone matrix),viruses, or bacterial cells.

An important aspect of the compositions and methods described herein isthat they can limit delivery of molecules to only selected (i.e.,selectable) portions of the body of the human to whom they areadministered. Many pharmaceutical agents, for example, exert beneficialeffects at some body locations, but exert deleterious effects at otherbody locations. Similarly, it is desirable to use some agents (e.g.,radionuclides and imaging agents) at the lowest dose that is practical,in order to minimize exposure of the human to the agent. The ability ofthe compositions described herein to direct delivery of such agents toparticular locations, structures, or cell types where they are to act isbeneficial.

The compositions and methods described herein have significant in vitroand in vivo uses stemming from the ability of the HSA and the fattyacid-complexed molecule to exhibit independent target-binding abilities.By way of example, a rHSA described herein can include a polypeptideportion inserted in place of a surface loop of HSA, wherein the portionconfers the ability to bind to a first target to the rHSA. A molecule(e.g., a polypeptide, a polynucleotide, or a small molecule such as acarbohydrate) capable of binding a second target can be conjugated witha fatty acid, and the fatty acid-conjugated molecule can be complexedwith the rHSA. Such pairs of rHSAs and fatty acid-conjugated moleculescan be used to bind a second target in the vicinity of a first target,to bind a first target in the vicinity of a second target, or toseparately bind first and second targets and subsequently (i.e., uponcomplexation) bring together the bound first and second targets. Askilled artisan recognizes that a wide variety of situations in whichsuch functionalities can be used, including purification, targeting,probing, binding, coating, and detection uses.

Particular aspects of the compositions and methods described herein aredescribed in greater detail below.

Conjugation of a Fatty Acid and a Molecule to be Complexed with a HSA

LCFAs (16-20 carbons) are essential for many cellular functions.Although the mechanisms of LCFA uptake by various cells are not fullyunderstood, non-esterified fatty acids are bound and transported byalbumins (the binding constant being from about 0.05 to 1 micromolar formyristic acids). HSA binding increases the effective aqueous solubilityof hydrophobic LCFAs to the millimolar range in blood and serum. One HSAmolecule is able to bind from about 0.1 to 2 molecules of LCFA undernormal physiological conditions and carries up to 6 molecules of LCFAafter exercise, fasting, adrenergic stimulation, and under certainpathological conditions. The crystal structures of complexes formedbetween LCFAs and HSA reveals at least 5 primary binding sites and 4putative binding sites. LCFA-conjugated molecules bind to HSA and thepharmacokinetic profiles (and corresponding therapeutic capacity) of themolecules are altered thereby. In each actual and putative binding sitein the HSA-LCFA complex, the carboxylate head-group of the LCFA isrigidly held in place by electrostatic interactions and hydrogen bonds,and the methylene tail is accommodated in a relative hydrophobic cavityof HSA.

Since the carboxylate head and the methylene end are located accessibleto the protein surface, it was hypothesized that chemical modificationsat these two ends would be tolerated without disrupting LCFA bindingwith HSA. For this reason, it is preferable to conjugate a molecule thatone wishes to deliver to a human cell at or near (i.e., within a fewmolecular bonds of) one end or the other of a fatty acid, and preferablyat or near an end of a LCFA. The interactions between HSA and the fattyacid serve to anchor the molecule to the HSA, by means of bindingbetween HSA and the fatty acid. The identity of the fatty acid used inthe compositions and methods described herein is not critical, becausethe ability of albumins such as HSA to bind with a variety of fattyacids is known. C₁₀-C₂₀ fatty acids (i.e., those which have from 10 to20 carbon atoms in their chemical structure are suitable, and C₁₆-C₂₀fatty acids may be particularly useful. The degree of branching andunsaturation are not critical, and straight chains fatty acids and thosehaving limited unsaturation are recognized as being among the fattyacids common in the diet and naturally occurring in animals such ashumans. Each of these fatty acids is suitable for use in thecompositions and methods described herein.

The chemical means or bonds by which the molecule is conjugated with thefatty acid are not critical. In one embodiment, the conjugation of themolecule and fatty acid is selected on the basis of being reversibleupon uptake of the conjugate within a cell (i.e., the conjugatinglinkage is physiologically reversible). By way of example, the reducingenvironment present in many cells can result in cleavage of a disulfidebond between a molecule and a thiol-substituted fatty acid, resulting ingeneration of free molecule and free fatty acid upon uptake of theconjugate into a cell. Physiological reversibility of the conjugation isnot necessary unless the conjugation prevents the molecule fromexhibiting its desired function. By contrast, conjugation that limits oreliminates a property of the molecule to be delivered can be beneficialfor preventing inappropriate exhibition of that property prior tocellular uptake.

In one embodiment, the fatty acid can be conjugated with a portion of apeptide that can be cleaved by an enzyme known to occur at (or suppliedto) a body location at which release of the fatty acid from the peptideis desired. When the complex described herein (or the fatty acidconjugate alone) encounters the enzyme, cleavage of the peptide occurswith release of the fatty acid from the cleaved portion of the peptide.

In another embodiment, the fatty acid can be conjugated with a peptideand selected to be a fatty acid moiety that is cleavable by a lipaseenzyme, such as a phospholipase. When the complex encounters the lipase,cleavage of the fatty acid moiety from the peptide moiety occurs.

By way of example, a method of conjugating a polynucleotide (e.g.,single- or double-stranded DNA or RNA) with a fatty acid are illustratedhere. The oligonucleotide can be first modified at the 5′- or 3′-endusing an amine carbon modifier, resulting in a molecule having thestructure NH₂—(CH₂)_(n)-polynucleotide. Examples of aminecarbon-modifying reagents are known in the art. These linkers can beadded to the 5′-end, to the 3′-end, or to both ends of thepolynucleotide. They can also be added to modified base residues withinthe polynucleotide by known methods. The amine-modified polynucleotideis conjugated with the carboxyl group of a fatty acid, forming an amidebond and resulting in a conjugate having the structureCH₃—(CH₂)_(m)—CONH—(CH₂)_(n)-polynucleotide, wherein m and n areindependently integers. The length of the fatty acid can be selected,using routine methods, for optimal HSA binding for different forms andlengths of polynucleotide. Similarly, a fatty acid and a polypeptide canbe conjugated by forming an amide bond using the fatty acid carboxylgroup and an amine group (i.e., an N-terminal amine or a primary amineside group, such as that of lysine) to yield the structureCH₃—(CH₂)_(m)—CONH-polypeptide. Likewise, employing an analogousconjugation strategy, other relatively small molecules can be conjugatedto the carboxyl end of fatty acids through amide bonds.

Further by way of example, a method of conjugating a polynucleotide(e.g., single-stranded DNA or RNA) with a fatty acid are illustratedhere. The oligonucleotide can be first modified at the 5′ or 3′ endusing a thiol carbon modifier (resulting in a molecule having thestructure HS—(CH₂)_(n)-polynucleotide). Examples of thiolcarbon-modifying reagents are known in the art. The thiol-modifiedoligonucleotide can be conjugated using the thiol group of 1-mercaptofatty acids (HS—(CH₂)_(m)—COOH) to form a disulfide bond, yielding aconjugate having the structurepolynucleotide-(CH₂)_(n)—S—S—(CH₂)_(m)—COOH. As with amide-conjugatedconjugates, the length of the fatty acid can be selected, using routinemethods, for optimal HSA binding for different forms and lengths ofpolynucleotide. Similarly, a fatty acid and a polypeptide can beconjugated by forming a disulfide bond using a thiol group of a cysteineamino acid of the polypeptide and a 1-mercapto fatty acid, to yield thestructure polypeptide-S—S—(CH₂)_(m)—COOH. Likewise, employing ananalogous conjugation strategy, other relatively small molecules can beconjugated to the methylene end of the fatty acid by way of, forexample, a disulfide bond.

Instead of, or in addition to, amine and thiol linkers, carboxyl linkerscan also be used to conjugate fatty acids (or C₁₀-C₂₀ hydrocarbons) to amolecule such as a polynucleotide (e.g., forming a structure having aformula like COOH—(CH₂)_(n)-polynucleotide). In this embodiment, acarboxyl linking group is attached to the polynucleotide using knownmethods. A primary amine having a C₁₀-C₂₀ hydrocarbon tail (i.e., acompound having a structure like CH₃—(CH₂)_(m)—NH₂) can thereafter beconjugated with the carboxyl moiety, yielding a primary amine moietylinked to the polynucleotide and having a fatty acid-like hydrocarbontail (i.e., a compound having a structure likeCH₃—(CH₂)_(m)—NHCO—(CH₂)_(n)-polynucleotide).

For example, a peptide can be conjugated with a fatty acid by way of acarboxyl, amine, or sulfhydryl group. Further by way of example, acarbohydrate can be conjugated with a fatty acid by way of a hydroxylgroup of the carbohydrate. Similarly, any molecule (including, forexample, a small pharmaceutically-active agent) can be conjugated with afatty acid by way of any of these, or other known reactive centers,using methods known in the art.

When conjugating molecules with a fatty acid, it is not critical thatall of the molecules or all of the fatty acids are conjugated followingthe conjugation reaction(s). However, if it undesirable to includenon-complexed, non-conjugated molecule in the composition to beadministered to a human, then it can be desirable to removenon-conjugated molecule, non-conjugated fatty acid, or both, followingthe conjugation reaction and prior to complexing the conjugate with theHSA. Alternatively, non-conjugated molecule can be separated fromHSA-complexed, fatty acid-conjugated molecule after the products of theconjugation reaction(s) are complexed with the HSA. Because the fattyacid use must be able to form a complex with the HSA, it may beimpractical to remove non-conjugated fatty acid from the compositionafter contacting the products of the conjugation reaction(s) arecomplexed with the HSA. For that reason, if complexation onnon-conjugated fatty acid is undesirable, then non-conjugated fattyacids should be separated from the products of the conjugationreaction(s) prior to contacting those products with the HSA, for exampleby reverse phase chromatography.

The identity of the molecule that is complexed with the fatty acid isnot critical. A wide variety of molecules can be conjugated with a fattyacid and the resulting conjugate can be complexed with a HSA. Formolecules intended to be delivered by way of a body fluid such as bloodor peritoneal fluid, the molecule should be selected such that thecomplex formed after conjugating the molecule with the fatty acid andcomplexation of that conjugate with the HSA exhibits at least minimalsolubility in the body fluid (in many instances, it is immaterialwhether the complex is dissolved or suspended in the fluid). Similarly,appropriate physical forms (e.g., suspensions and solutions) of thecomplex are apparent to the skilled artisan for administration,depending on the identity of the route of intended administration andthe corresponding body fluid our surface. The methods described hereincan be used even with completely (or nearly completely) insolublemolecules and materials. By way of example, a plastic material (variouspolymeric materials, including, e.g., the housing of an implantabledevice) can be conjugated with a fatty acid, and the conjugated materialcan be contacted with a HSA prior to implanting the device, to minimizeimmunogenicity and binding of proteins other than HSA with the materialfollowing implantation.

The identity of the molecule to be conjugated with a fatty acid andcomplexed with an albumin is not critical. Although it is foreseeablethat there will be an upper limit on the size (e.g., as assessed bymolecular weight or number of monomeric units) of the molecule that canbe effectively conjugated and complexed, a skilled artisan understandsthat the upper limit, if any, can be determined empirically, and suchdetermination involves no more than the exercise of normal experimentaltechnique. By way of guidance, double-stranded oligonucleotides having alength of about 30 base pairs can be conjugated and complexed, and thosehaving a length not greater than 22 base pairs appear to be particularlyamenable to conjugation and complexation using the methods describedherein. Similarly, polypeptides having a length of about 40 amino acidresidues can be conjugated and complexed, and those having a length notgreater than 20 residues appear to be particularly amenable toconjugation and complexation using the methods described herein. Therelative hydrophobicity and hydrophilicity of the compound to beconjugated and complexed do not appear to be of particular importance.

It is not critical that the molecule be conjugated with one (i.e., andonly one) species of fatty acid. The molecule can be conjugated with amixture of fatty acids (e.g., a mixture of C₁₀-C₂₀ fatty acids) yieldingconjugates in which the molecule-moiety is identical among theconjugates, but in which the fatty acid moieties vary among theconjugates. Likewise, if conjugation with substantially only a singlefatty acid species is desired, it is not critical that the fatty acid befree of all other fatty acids. Either synthetic (i.e.,non-biologically-synthesized) fatty acids or fatty acids obtained from abiological source can be used, it being recognized that fatty acidsobtained from natural sources often contain a mixture of fatty acidspecies.

Details of reagents and reaction conditions not specifically describedherein are readily determinable by a skilled artisan in this field.

Complexing HSA with the Fatty Acid-Conjugated Molecule

The target-binding albumins described herein can be used on their own,without a fatty acid-conjugated molecule complexed therewith. Inimportant embodiments of the methods and compositions described herein,a fatty acid-conjugated molecule is present in a complex wherein atleast the fatty acid portion of the conjugate interacts with an HSAprotein described herein, such as an rHSA or one of the target-bindingalbumins described herein. This complex is formed by contacting theconjugate with a composition that includes HSA. The conjugate and theHSA-containing composition are contacted for a time and under conditionssufficient for the conjugate to bind with the albumin to form a complex.

The duration and conditions under which the compositions need becontacted depend on the particular properties of the molecule, the fattyacid, and the HSA that are used, and these duration and conditionparameters are readily determinable by a skilled artisan in this field.By way of example, when the first molecule is a polypeptide having thesequence CHLDAHWKG (SEQ ID NO: 5) and is conjugated with the fatty acidderivative 16-methanethiolsulfonyl hexadecanoic acid by way of the thiolgroup of the cysteine residue of the polypeptide, and that conjugate iscontacted with naturally-occurring HSA in a phosphate-buffered salinesolution at 20 degrees Celsius, complexation of the conjugate and theHSA will normally be essentially complete within about 30 minutes. Askilled artisan in this field recognizes that the reagents and reactiontimes that are appropriate depend on the chemical characteristics of thefatty acid and molecule-to-be-conjugated species. If not predictablefrom the identity of the species, the skilled artisan understands thatthey can be determined empirically using no more than ordinaryexperimentation. The HSA with which the conjugate is to be complexed ispreferably present in excess (e.g., 2- to 5-fold molar excess), and theprecise ratio of conjugate to HSA is not critical. The time required forapproximately equilibrium binding between the HSA and the conjugate isdependent on the chemical species involved, the temperature, the degreeand type of agitation, and other factors readily known to the skilledartisan. The duration for which complex formation is allotted is notcritical and should generally be on the time scale of minutes to hours(typically 30 minutes to 12 hours, with preference for durations lessthan several hours).

The HSA can be a naturally-occurring HSA or a synthetic HSA protein.Synthetic HSA proteins can have the same primary amino acid structure asnaturally-occurring HSA, or they can be engineered to have a differentamino acid structure (so long as normal HSA activities are substantiallypreserved; i.e., so long as the HSA exhibits the biochemical andbiological properties of HSA). HSA and the recombinant HSAs describedherein must be used in human in vivo applications. For in vivoapplications in another animal, an albumin that occurs naturally in thatother animal must be used, and non-human animal albumins can becomplexed with fatty acid-conjugated molecules just as described hereinfor HSA. For in vitro application, the immune response of an animalagainst the albumin of a different animal need not be considered, andsubstantially any albumin can be used. Thus, while the compositions andmethods described herein have been discussed primarily in the context ofusing HSA, substantially the same compositions and methods can be madeand performed for in vitro uses using albumins obtained from otheranimals, such as bovine serum albumin.

HSA can obtained from a natural source (e.g., from blood or plasmaharvested from a human donor). HSA can also be obtained from an organism(e.g., a bacterium, a fungus, a plant cell culture, or an insect cellculture) that does not normally produce HSA, but has been engineered todo so. A characteristic of HSA obtained from natural sources is that theHSA so obtained can bind with fatty acids that occur in the organism orthe medium in which it is produced. HSA saturated with fatty acids orlipids can exhibit a limited capacity to form a complex with the fattyacid-molecule complexes described herein. If the HSA with which acomplex is to be formed has been obtained from a natural source, then itcan be desirable to remove some or all of the fatty acids and lipidsthat are associated with the HSA prior to contacting the HSA with thefatty acid-molecule conjugate described herein. Such a process iscommonly referred to as “defatting” the HSA, and substantially any knowndefatting procedure can be used. An example of a suitable defattingprocedure (one involving charcoal treatment) is described in Chen, 1967,J. Biol. Chem. 242:173-181.

A preparation of HSA obtained from a natural source can include ascontaminants other molecules that naturally occur in the source. Forexample, HSA preparations obtained from an organism that produces theHSA can be contaminated with other proteins normally produced by theorganism. Furthermore, if the HSA is obtained from a human, the HSApreparation can include as a contaminant one or more human pathogens.Thus, although human blood and other human body fluids can be convenientand economical sources of HSA, the risk of pathogenic contamination bythe fluid donor can make HSA obtained from non-human sources (e.g.,cultured plant or insect cells) a preferable reagent in the compositionsand methods described herein.

The conditions under which and apparatus with which the compositionincluding the HSA and the composition including the fatty acid-moleculeconjugate are contacted are not critical. The goal of such contacting isto bring the conjugate into sufficient proximity to the HSA that theforces of molecular attraction between the HSA and the fatty acidportion of the conjugate are able to act and cause complex formation. Ifthe HSA and the conjugate are both soluble in a solvent that does notsignificantly denature the HSA, then the HSA and the conjugate cansimply be combined in the solvent, and stirring or other agitation neednot be applied. If the conjugate is in a solvent that is substantiallyinsoluble with the solvent in which the HSA is suspended, then aninterface will exist between the two solvents, and complexation canoccur substantially only at the interface. In such instances, proceduresfor contacting insoluble liquid phases (e.g., stirring, shaking, mixing,or emulsification) can be performed in order to increase the rate ofcomplex formation. If such procedures are employed, care should be takento avoid denaturing or otherwise damaging the HSA.

Considerations of appropriate liquid contacting procedures are wellknown in the art and a skilled artisan can select appropriate conditionswithout significant experimentation by taking into account the identityand nature of the solvents, the properties of the HSA and the conjugate,the temperature, the type and degree of agitation, and other factorswithin the ken of the skilled artisan.

An HSA protein can form a complex with multiple copies of themolecule-fatty acid conjugate. For example, if the HSA is not complexedwith any other lipid or fatty acid, naturally-occurring HSA can form acomplex with at least five conjugates. Several other putative fattyacid-binding sites are recognized in HSA, so it is possible that morethan 5 (e.g., 10, 12, or 14) conjugates can be complexed with a singleHSA molecule. With these guidelines in mind, the ratio of HSA moleculesto conjugates that are contacted is not critical. It is not necessarythat every fatty acid-binding site of the HSA be occupied with aconjugate molecule. It can be desirable to include as many conjugates inthe complex as possible, and this can be achieved by contacting anexcess (e.g., 20-fold molecular excess) of the conjugate with the HSAand permitting the complexation to proceed essentially to equilibrium.Appropriate HSA-to-conjugate ratios can be determined empirically aswell.

Complexes containing more than one type of fatty acid-molecule conjugatecan be formed by contacting the HSA with multiple types of conjugates.The contacting can be performed in multiple steps (e.g., contacting HSAwith a first conjugate and thereafter with a second conjugate) or bycontacting the HSA with a composition that includes multiple conjugates.The conjugates can be conjugates of a single fatty acid with differentmolecules. Alternatively, the same molecule can be conjugated withdifferent fatty acids. The mixture of conjugates can include conjugatesthat vary both in their fatty acid moiety and their other-moleculemoiety. Such mixed conjugates/complexes can be used, for example, ascombination therapy whereby two drug moieties can be targeted to asingle cell type, each drug affecting a different biochemical pathway ortarget in the cell. Such complexes can also include oneconjugated-molecule that tends to favor targeting of selected cells bythe complex, while another conjugated-molecule of the complex has adesired biological effect on the targeted cell. By way of example, acomplex can include a fatty acid-conjugated ligand of a cell surfacereceptor expressed on a desired cell type for the purpose of targetingthe complex to the desired cells, and the complex can further include afatty acid-conjugated moiety (e.g., a polypeptide or an oligonucleotide)for the purpose of delivering the moiety to the desired cells.

In one embodiment, the HSA is contacted with a second conjugate inaddition to the fatty acid-molecule conjugate. The second conjugate is asecond fatty acid moiety (i.e., either the same fatty acid moiety as inthe fatty acid-molecule conjugate or a different fatty acid moiety)conjugated with a compound that binds specifically with a targetmolecule that occurs on the surface of a cell to which the compositionis to be targeted or a target molecule that occurs in a tissue to whichthe composition is to be targeted. In these embodiments, it isinteraction between the second conjugate and the target that directs thecomplex (including the molecule to be delivered) to the target.

In another embodiment, the HSA is contacted with a second compound thatforms a complex with HSA, but is not a fatty acid-conjugated molecule.Numerous compounds (including many drugs, especially those havingsignificant hydrophobic portions) are known to form complexes with HSAwhen the compound is contacted with HSA. Such compounds can be complexedwith an HSA described herein (e.g., a rHSA or a target-binding albumin),either in vitro or in vivo. The complex can also be contacted with afatty acid-molecule conjugate of one of the types described herein,thereby forming a complex that includes the HSA, the compound, and theconjugate. Formation of the complex in vitro and separation therefrom ofnon-complexed components can yield complex having greater homogeneityand a more controlled composition than would be formed if the componentswere separately administered to an animal or to another system. Infields such as drug delivery, homogeneity (or at least partial control)of complex structure can be beneficial.

Complexation of the HSA with the conjugate preferably occurs after theconjugation reaction is complete or substantially complete. Although thecomplexation can be performed simultaneously with conjugation,performance of the two reactions simultaneously can result inconjugation of HSA with the fatty acid, with the molecule, with otherHSA molecules, or with some combination of these.

Purification

The compositions described herein can be purified to remove undesirablecomponents at various stages, using any of the wide variety ofpurification technologies known in the art. By way of example, it can beundesirable to have reagents employed in the fatty acid-moleculeconjugation reaction present when the conjugate is complexed with HSA(i.e., because conjugation of the conjugate or other components to HSAis undesirable). It can be preferable, therefore, to purify theconjugate from such reagents. The term “purify” does not refer to purityin a philosophical sense (i.e., utter absence of the non-desiredspecies), but instead means reduction of the concentration ofnon-desired molecules to levels at which the magnitude of theirundesirable effects is acceptable. Acceptable degrees of purity arereadily determinable by a skilled artisan in view of the disclosureprovided herein.

Formation of the fatty acid-conjugated, HSA-complexed molecular speciesdescribed herein can be desired in order to avoid an undesirableproperty or effect of the non-conjugated, non-complexed molecularspecies. As with substantially all chemical reactions, the conjugationand complexation reactions described herein cannot be expected to yieldhomogenous compositions which include the fatty acid-conjugated,HSA-complexed molecular species in the complete absence ofnon-conjugated and/or non-complexed species. If undesirable propertiesof those species are to be avoided upon administration of thecomposition to a human, it can be desirable to separate thosenon-conjugated and/or non-complexed species from the composition priorto administration. Any of the wide variety of purification procedures(e.g., chromatography, electrophoresis, centrifugation) known in the artcan be used to effect such purification. A quick, simple, and effectiveway of separating relatively large molecules from substantially smallermolecules is ultrafiltration, in which pressure is applied to a liquidmixture above a membrane having pores of sufficient size tosubstantially retard passage of the large molecules while permittingpassage of small molecules. Ultrafiltration methods can be used topurify products described herein from substantially smaller molecularspecies (e.g., from conjugation reagents or from non-conjugated,non-complexed drug molecules).

Recombinant HSA (rHSA) Proteins

Chemically-modified HSAs, monoclonal antibodies, ligand- orpeptide-conjugated polymers, and liposomes have been applied by othersto achieve drug targeting and delivery. However, availability,biocompatibility, toxicity, durability and immunogenicity have limitedthe clinical and other application of those approaches. By contrast, thetechnology described herein can be employed to form a complex of HSAwith a fatty acid conjugate of such compounds, such complexes optionallyincluding other components, as described herein.

In an important embodiment, the HSA that is used in the methodsdescribed herein can be a rHSA that exhibits a molecular or cellulartargeting function. Design and production of recombinant proteins hasbecome routine, and a skilled artisan in this field is able to constructa wide variety of rHSAs in view of the information described herein andthe information available in the art. In contrast to prior descriptions,this disclosure emphasizes the importance of inserting a polypeptidesequence that confers a molecular or cellular targeting function inplace of a disulfide-bounded surface loop of HSA.

The HSAs (including the corresponding recombinant animal albumins)described herein have a polypeptide sequence that replaces a surfaceloop of the native albumin (i.e., it replaces one of the surface loopsof HSA described herein or an equivalent loop of a corresponding animalalbumin). Occurrence of disulfide bond bounding the polypeptide sequencethat replaces a surface loop of HSA is significant, in that the presenceof the disulfide bond tends to limit both the effect of the insertedsequence on the normal structure (and the corresponding biochemical andbiological functions) of HSA and the effect of non-replaced HSA proteinconformation on the structure (and the corresponding biochemical andbiological functions) of the polypeptide sequence that replaces thesurface loop of the HSA. In this way, the rHSA proteins described hereincan be considered to have a modular structure, wherein the rHSA retainsmost of the modules (especially the non-surface modules) ofnaturally-occurring HSA and also includes one or more modules (havingmolecular- or cellular-targeting function, for example) on the surfaceof the HSA. The rHSA proteins thereby retain the fatty acid-complexingand other normal biochemical and biological properties of HSA, whilealso exhibiting the properties (e.g., cell- or molecule-targeting)properties of the module(s) installed in place of an HSA surface loop.

An rHSA that exhibits a molecular or cellular targeting function is madeby exchanging a functional polypeptide sequence (e.g., a fragment of aprotein that mediates an interaction between a protein and either atarget protein or some other target molecule) in place of a surface loopof HSA. Manufacture of fusion proteins is well known in the art. In thecontext of the HSA-containing complexes described herein, the fusionprotein should be a protein that retains substantially all of theprimary amino acid sequence of HSA and includes inserted polypeptidesequences in place of a surface loop of HSA.

An appropriate rHSA is created by inserting a functional peptidesequence in place of one or multiple surface loops of HSA. Those surfaceloops have been identified by others based on the crystal structure ofHSA, and are illustrated diagrammatically in FIG. 2. The rHSA possessesan engineered exogenous polypeptide region that exhibits a specificbinding function. Because that polypeptide region (or regions) isinserted only in place of one or more surface loops, the rHSA retainsthe native functions of HSA. Retention of the normal functions of HSA,as well as the normal non-immunogenicity of HSA makes the complexdescribed herein highly desirable as a delivery vector.

An interesting feature of HSA is that none of the 17 disulfide bonds ofnaturally-occurring HSA extend across the three domains (I, II, and IIIin FIG. 2) of HSA, and all of those disulfide bonds hold adjacentalpha-helical sequences in place. This kind of folding not onlystabilizes the protein but also offers a great degree of flexibility toHSA. These disulfide bonds also hold ribbon like loops that serve linkalpha-helixes. The loops that are present on the surface of the proteinare appropriate sites into which exogenous polypeptide sequences can beinserted, as illustrated in FIG. 3.

Because the ends of the HSA surface loops are held in place by disulfidebonds, the loops remain relative stable, even when an exogenouspolypeptide sequence is inserted in place of the surface loop thatnormally occurs between the disulfide-linked cysteine residues.Potential conformational changes attributable to polypeptide sequenceinserted within a surface loop are constrained locally by the disulfidebonds, and likely do not disturb nearby structures. It is likely forthat reason that normal HSA function remains undisturbed. It can beadvantageous to incorporate a polypeptide domain that has asubstantially looped secondary structure or a cyclic primary structure(i.e., in the native protein or screened peptide from which thepolypeptide sequence is derived) in place of a surface loop of HSA. Suchdomains, which retain more nearly their ‘naturally-occurring’ shape, aremore likely to retain the native or screened function than aresubstantially linear polypeptides which must be bent or strained to beincorporated in place of the loop. For a polypeptide region believed tohave a substantially linear conformation in its functional state orbelieved to have a secondary structure more complicated than the surfaceloop that it replaces, it can be advantageous to include a polypeptide‘linker’ region (e.g., one to several glycine residues on one or bothends of the polypeptide sequence) in order to confer sufficientflexibility to the inserted polypeptide that it can take up itsfunctional conformation. Design and insertion of such ‘linker’ regionsis well known in the art.

It is not critical which of the HSA surface loops is used forreplacement by the functional peptide sequence. The sequence can beinserted in place of one or more of the loops, and the same sequence ordifferent functional peptide sequences can be inserted in place ofdifferent surface loops of HSA. The surface loops of HSA are delineatedby the disulfide bridges that occur in naturally occurring HSA betweenresidues 53-62, 75-91, 90-101, 168-177, 245-253, 265-279, 278-289,360-369, 437-448, 461-477, 476-487, and 558-567 of SEQ ID NO: 1.

The format of sequence that will replace loops held by disulfide bondsis as follows.

For the surface loop represented by HSA residues 53-62:53Cys-Xaa_(n)-62Cys.

For the surface loop represented by HSA residues 75-91:75Cys-Xaa_(n)-90Cys-91Cys.

For the surface loop represented by HSA residues 90-101:90Cys-91Cys-Xaa_(n)-101Cys.

For the surface loop represented by HSA residues 168-177:168Cys169Cys-Xaa_(n)-177Cys.

For the surface loop represented by HSA residues 245-253:245Cys246Cys-Xaa_(n)-253Cys.

For the surface loop represented by HSA residues 265-279:265Cys-Xaa_(n)-278Cys279Cys.

For the surface loop represented by HSA residues 278-289:278Cys279Cys-Xaa_(n)-289Cys.

For the surface loop represented by HSA residues 360-369:360Cys361Cys-Xaa_(n)-369Cys.

For the surface loop represented by HSA residues 437-448:437Cys438Cys-Xaa_(n)-448Cys.

For the surface loop represented by HSA residues 461-477:461Cys-Xaa_(n)-476Cys477Cys.

For the surface loop represented by HSA residues 476-487:476Cys477Cys-Xaa_(n)-487Cys.

For the surface loop represented by HSA residues 558-567:558Cys559Cys-Xaa_(n)-567Cys.

In these formulae, Xaa represents any amino acid residue, and n can befrom 5 to about 30 (and is preferably from 7 to 12).

With regard to functional polypeptide sequences that can be insertedinto the surface loops, disulfide bonds are required to maintain andmimic proper conformation of some targeting sequences such as the RGDmotif In the RGD motif, a local disulfide link maintains the potentprotein binding ability of the motif When selecting an appropriateexogenous functional polypeptide sequence for replacement of a surfaceloop, the presence of a similar function-stabilizing disulfide bond ishighly desirable.

The RGD motif is clearly not the only polypeptide domain that can beinserted in place of a surface loop of HSA. Any of a wide variety ofknown polypeptide domains can be inserted, so long as the size of thedomain does not exceed a length of about 30 residues, and preferably notmore than about 25 or 12 residues. Examples of suitable protein domainsinclude the adrenomedulin domain (CRFGTC; SEQ ID NO: 6), theendothelin-1 domain (CSCSSLMDKE CVYFC; SEQ ID NO: 7), the matrixmetalloproteinase 9 (MMP9) binding peptide domain (CRRHWGFEFC; SEQ IDNO: 8), the matrix metalloproteinase 2 (MMP2) binding peptide domain(CTTHWGFTLC; SEQ ID NO: 9), and aminopeptidase N binding peptide domains(CNGRCVSGCA GRC and CVCNGRMEC; SEQ ID NOs: 10 and 11, respectively)Protein domains bounded by disulfide-linked cysteine residues areparticularly well-suited for insertion in place of a surface domain ofHSA.

As described herein, synthetic polypeptides can be generated in the formof libraries of random or semi-random sequences, and it is beneficial ifthe function of such polypeptide domains is screened in a system inwhich the domain occurs between disulfide-linked cysteine residues onthe surface of a protein, capsid, or other particle. A screenedpolypeptide sequence that exhibits a desirable function can be insertedin place of a surface loop of an HSA described herein.

The source of the functional polypeptide sequence inserted in place ofthe surface loop is not critical, so long as the appropriate targetingfunction of the inserted sequence can be realized. Appropriate sequencescan be derived from well-studied proteins (e.g., like theintegrin-binding RDG-motif of fibronectin). Such sequences can also bedetermined empirically, such as with sequences derived from a library ofcompounds can be screened for a particular target (e.g., a protein,peptide, nucleic acid, carbohydrate, or lipid). By way of example, aphage peptide display library can be constructed using known methods,wherein each phage of the library exhibits a polypeptide sequencebounded by a linked pair of cysteine residues. By screening such alibrary for polypeptides that exhibit a desired function (e.g., abilityto bind to a selected molecular target), a sequence suitable forconferring that property to an rHSA protein can be identified andinserted in place of an HSA surface loop as described herein. A skilledartisan appreciates that further refinement of the inserted sequence canbe performed by selective mutagenesis (coupled with screening for thedesired function) of the sequence inserted in place of the HSA surfaceloop.

The surface location of the HSA surface loops is necessary in order forthe exogenous functional polypeptide sequence to exert the targetingfunction that is desirable in the compositions described herein. Inorder to enhance the targeting ability, a high affinity bindingexogenous sequence and an optimal mutagenesis site of HSA can beselected, for example by screening of randomly or selectively mutatedclones of a polypeptide selected from among a library of cyclicpolypeptide domains. Such selective mutation can include, for example,replacement of amino acid residues of the polypeptide selected from thelibrary with similar amino acid residues. Furthermore, screening of rHSAspecies wherein one or a few residues of the retained HSA sequence ofthe rHSA that are adjacent (i.e., not within the surface loop and withinabout 5 residues) of the disulfide-linked cysteine residues that definethe surface loop can also improve the function, for example by improvingthe local protein conformation in a manner beneficial to the function ofthe inserted polypeptide domain. Alternatively, multiple targetingsequences can be into one or more of the surface loops of HSA. A skilledartisan in this field recognizes that these manipulations are inherentlyempirical, yet do not require more than ordinary experimentation.

The RGD motif (Arg-Gly-Asp) is an exhaustively-studied protein motifthat occurs in many extracellular matrix (ECM) proteins such asfibronectin and vitronectin. The RGD motif is recognized by integrins, afamily of heterodimeric transmembrane receptors. The alpha-v-beta-3integrin, which has an essential role in tumor angiogenesis andmetastasis, has been targeted by a variety of drug delivery vehicles.

The fibronectin RGD sequence (CGRGDSPC; SEQ ID NO: 4) was inserted intoHSA, replacing amino acid residues 53-62 of naturally-occurring HSA, asshown in FIGS. 3A and 3C. This fragment occurs with domain I of HSA andis located on the protein surface. Based on HSA structure andbiochemical studies, these residues are not expected to have anessential role in ligand binding or protein folding. Therefore,mutations in this region should be well tolerated.

We cloned, expressed and purified this rHSA from a Pichia pastoris yeastrHSA expression system. The protein was soluble and was purified toapparent homogeneity. We confirmed the identity of the purified proteinby Western blotting using anti-HSA antibody and anti-c-Myc antibody. Thealpha-v-beta-3 integrin binding ability of RGD-HSA was confirmed byco-immunoprecipitation and Western blotting.

The HSA proteins described herein, having a polypeptide inserted inplace of a surface loop of native HSA, can be used alone, in a form inwhich one or more molecules (e.g., a detectable marker or an affinitytag) is conjugated therewith, in a form in which the HSA is complexedwith a fatty acid-conjugated molecule, in a form in which the HSA iscomplexed with a compound other than a fatty acid-conjugated molecule,or in a combination of these.

Examples

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations which are evident as a result of the teaching providedherein.

Cloning strategy to construct a Human Serum Albumin cloning vector toreplace a specific loop with a functional peptide.

This cloning strategy is described with reference to FIG. 4. Tworestriction enzyme cutting sites (A and B) close to the two cysteineresidues that form a disulfide bond and hold a surface loop structureare designed and selected by changing the nucleotide coding sequence ofthe amino acid residues of the loop. For most cases, the generation ofcloning sites A and B will only change the amino acid coding sequencebut not the identity of the encoded amino acid residue. If the aminoacid and its coding sequence have to be changed to generate a pair ofproper cloning sites, a relative conserved amino acid at this positionis selected to minimize perturbation of enzymatic activities of themutant albumin. In addition, the cloning sites A and B can be selectedat the positions of cysteine residues or either outside or inside of theloop. It is critical that these two sites must not cut the albumin geneand the cloning vector in such a way that expression of the balance ofthe albumin sequence is lost.

Once the cloning sites are determined, the following steps will beperformed to construct the HSA cloning vector. The cloning strategy canbe done using, for example, pPICZαA, B, C (INVITROGEN), pET21a, b, c(NOVAGEN) or other cloning vectors. Step 1). A gene fragment (I) of HSAthat includes the DNA sequence encoding the amino-terminus of HSAthrough the first cysteine residue of the surface loop to be replaced isamplified by PCR. The 5′ primer contains a cloning site of the vectorclose to 5′ end of the multiple cloning sites region. The 3′ primerincludes the restriction enzyme cutting sites A and B and a cloning siteof the vector close to the 3′ end of the multiple cloning sites region.Step 2) The vector plasmid is cut with selected restriction enzymes tocreate the 5′ and 3′ cloning sites. Step 3) The fragment I from step 1is ligated into the linearized vector. Step 4) The circular vector fromstep 3 is cut with selected restriction enzymes to create the B and 3′cloning sites. Step 5) A gene fragment (II) of HSA that includes the DNAsequence encoding the second cysteine residue of the surface loop to bereplaced through the carboxyl-terminus of the HSA is amplified by PCR. Astop codon can be included at the end of HSA gene, or a DNA linker canbe included at the end of HSA gene to add a peptide tag fragment (e.g.,His₆ tag, c-Myc tag etc.) or construct fusion proteins. Step 6) Thefragment II from step 5 is ligated into the linearized vector in step 4.A DNA sequence that encodes a functional peptide sequence can besynthesized and cloned into the A and B cloning sites of the vectorobtained in step 6.

FIG. 5 shows an arrangement of PCR primers useful in the construction ofan expression vector for generating a recombinant human albumin usingpPICZαA vector (INVITROGEN). In the figure, replacement of a specificalbumin loop with a functional peptide is achieved as follows. Twocloning sites encompassing the loop to be replaced are created in thehuman serum albumin gene. Using these two universal modules, a varietyof peptides can be inserted between the two cysteines and form a surfaceloop. To create these two cloning sites, the HSA gene is divided intotwo fragments, i) HSA Fragment I, which ranges from the 5′ end of thegene to the position of the first cysteine residue of the loop and ii)HSA Fragment II, which ranges from the cloning site near the secondcysteine residue of the loop to the 3′ end of the gene. In FIG. 5, FRI5′and FRI3′ stand for the 5′ and 3′ primers used to amplify (by PCR) theHSA Fragment I, respectively. The FRII5′ and FRII3′ stand for the 5′ and3′ primers used to amplify the HAS Fragment II, respectively. For allloops, the common FRI5′ and FRII3′ primers can be used. The FRI3′ andFRII5′ are determined based on the location of the loop, as shown inFIGS. 6-17.

FIG. 6A shows the sequence (SEQ ID NO: 12) of the FRI5′ primer. ThepPICZαA vector is for the secreting expression of protein. The Xho Icloning site is used to generate the 5′ sticky end DNA. For the purposeof construction of a N-terminal albumin fusion protein using thisvector, two cloning sites, EcoR I and Pml I can be included between theSte 12 cleavage site and the 5′ of HSA gene. This variant is shown inFIG. 6B.

Appropriate sequences for FRII3′ primers are shown in FIG. 7. The Sac IIcloning site is chosen as the 3′ end cloning site. Other cloning sitessuch as Not I, Xba I and Sal I can also be selected for the cloningpurpose, as shown in FIG. 7A. To obtain a targeting albumin with aC-terminal tag (c-Myc and His6) attached to the pPICZαA vector, a primeras shown in FIG. 7B can be used. To obtain a targeting albumin with acloning site for a C-terminal albumin fusion protein, a primer as shownin FIG. 7C can be used.

Primers corresponding to the loop between residues 53-62 of HSA areshown in FIG. 8. The primer shown in FIG. 8A is a FRI3′ primer, and theFsp I cloning site changes the C53V54 to C53A54 (i.e., cysteine residueat position 53 and valine residue at position 54 to a cysteine residueat position 53 and alanine residue at position 54; similar terminologyis used herein without this detailed explanation in each instance, thesingle-letter amino acid codes being well known). The primer shown inFIG. 8B is a FRII5′-1 primer, and the Sph I cloning site changes theN61062 to A61062. The primer shown in FIG. 8C is a FRII5′-2 primer, andthe Hind III cloning site changes the coding sequence of K73L74 from AAATAA (SEQ ID NO: 20) to AAG CTT (SEQ ID NO: 21).

Primers corresponding to the loop between residues 75-90 of HSA areshown in FIG. 9. The primer shown in FIG. 9A is a FRI3′ primer, and theFsp I cloning site changes the C75V76 to C75A76. The primer shown inFIG. 9B is a FRII5′-1 primer, and the Sph I cloning site changes theD90C91 to A90C91. The primer shown in FIG. 9C is a FRII5′-2 primer, andthe Nhe I cloning site changes the A92K93 to A92S93.

Primers corresponding to the loop between residues 91-101 of HSA areshown in FIG. 10. The primer shown in FIG. 10A is a FRI3′ primer, andthe Fsp I cloning site changes the coding sequence of C91A93 from TGTGCA (SEQ ID NO: 28) to TGC GCA (SEQ ID NO: 29). The primer shown in FIG.10B is a FRII5′-1 primer, and the Sph I cloning site changes theE100C101 to A100C101. The primer shown in FIG. 10C is a FRII5′-2 primer,and the Hind III cloning site changes F102L103 TO K102L103.

Primers corresponding to the loop between residues 169-177 of HSA areshown in FIG. 11. The primer shown in FIG. 11A is a FRI3′ primer, andthe Fsp I cloning site changes the C169Q170 TO C169A170. The primershown in FIG. 11B is a FRII5′-1 primer, and the Sph I cloning sitechanges the coding sequence of A176C177 from GCC TGC (SEQ ID NO: 30) toGCA TGC (SEQ ID NO: 31). The primer shown in FIG. 11C is a FRII5′-2primer, and the Hind III cloning site changes coding sequence ofK181L182 from AAG CTC (SEQ ID NO: 32) to AAG CTT (SEQ ID NO: 33).

Primers corresponding to the loop between residues 265-278 of HSA areshown in FIG. 12. The primer shown in FIG. 12A is a FRI3′ primer, andthe Fsp I cloning site changes the C265E266 to C265A266. The primershown in FIG. 12B is a FRII5′-1 primer, and the Sph I cloning sitechanges the E277C278 to A277C278. The primer shown in FIG. 12C is aFRII5′-2 primer, and the Nhe I cloning site changes the K286S287 toA286S287.

Primers corresponding to the loop between residues 279-289 of HSA areshown in FIG. 13. The primer shown in FIG. 13A is a FRI3′ primer, andthe Fsp I cloning site changes the C279E280 to C279A280. The primershown in FIG. 13B is a FRII5′-1 primer, and the Sph I cloning sitechanges the H288C289 to A288C289. The primer shown in FIG. 13C is aFRII5′-2 primer, and the Nhe I cloning site changes the A291E292 to A291S292.

Primers corresponding to the loop between residues 361-369 of HSA areshown in FIG. 14. The primer shown in FIG. 14A is a FRI3′ primer, andthe Fsp I cloning site changes the coding sequence of C361A362 fromTGTGCC (SEQ ID NO: 46) to TGCGCA (SEQ ID NO: 47). The primer shown inFIG. 14B is a FRII5′-1 primer, and the Sph I cloning site changes theE368C369 to A368C369. The primer shown in FIG. 14C is a FRII5′-2 primer,and the Nhe I cloning site changes the A371K372 to A371S372.

Primers corresponding to the loop between residues 438-448 of HSA areshown in FIG. 15. The primer shown in FIG. 15A is a FRI3′ primer, andthe Fsp I cloning site changes the C438K439 to C438A439. The primershown in FIG. 15B is a FRII5′-1 primer, and the Sph I cloning sitechanges the P447C448 to A447C448. The primer shown in FIG. 15C is aFRII5′-2 primer, and the Nhe I cloning site changes the L453S454 toA453S454.

Primers corresponding to the loop between residues 461-467 of HSA areshown in FIG. 16. The primer shown in FIG. 16A is a FRI3′ primer, andthe Fsp I cloning site changes the C461V462 to C461A462. The primershown in FIG. 16B is a FRII5′-1 primer, and the Sph I cloning sitechanges the K475C476 to A475C476. The primer shown in FIG. 16C is aFRII5′-2 primer, and the Hind III cloning site changes the codingsequence of E478S479L480 from GAATCCTTG (SEQ ID NO: 52) to GAAAGCTTG(SEQ ID NO: 53).

A primer and a HSA II fragment corresponding to the loop betweenresidues 559-567 of HSA are shown in FIG. 17. The primer shown in FIG.17A is a FRI3′ primer, and the Fsp I cloning site changes the 559C560Kto 559C560A. Shown in FIG. 17B is an HSA fragment useful for makingrHSAs having this loop replaced by another polypeptide.

If the Hind III site is used to construct the HSA fragment II, the HindIII site of pPICZαA at the position of 872-877 is eliminated first. Thatis, 871 CAAGCTTGTT 880 (SEQ ID NO: 56) is changed to 871 CAAGCATGTT 880(SEQ ID NO: 57) by site-directed mutagenesis.

Because loops located between residues 124-169, 200-246, 245-253,316-361, 392-438, 467-487, and 514-559 either are not exposed to thesurface or are too long to form a simple structure, these loops are notconsidered preferable candidates to swap with a functional peptide.However, if a domain of albumin instead of a whole protein is used toconstruct a targeting protein, these loops may become surface loops.Therefore, similar strategy can be applied to replace these loops with afunctional peptide.

The primers and cloning strategies described in this section aredesigned based on the pPICZαA cloning vector. These strategies are alsoapplicable to other cloning vectors. The cloning sites encompassing adesignated loop are selected based on the multiple cloning sites of avector.

It was demonstrated that recombinant wild type HSA and RGD-containingHSAs (exhibit comparable fatty acids binding abilities. BODIPY fattyacids (Invitrogen, D3823, 20 pmol) were incubated at 20 C with 0.2 or 5micrograms of HSA in 10 μl buffer with 20 mM Tris-HCl (pH 6.8) and 150mM NaCl for 15 minutes. The mixtures were then added with 3 μl ofloading buffer (20 mM Tris-HCl (pH 6.8) and 150 mM NaCl with 50%sucrose) and loaded to 1×TBE acrylamide gel. The products were separatedunder 12 mA for 45 minutes. The gel was visualized under 352 nm UV.Comparable degrees of fatty acid binding were observed.

Recombinant wild type HSA and RGD-containing HSAs (HSA-FN and HSA-4C,respectively) were shown to exhibit comparable esterase activities. Thereaction of p-nitrophenyl acetate hydrolysis-mediated by rHSA andRGD-rHSA was assessed spectrophotometrically at 390 nm by monitoring theappearance of p-nitrophenol. The reaction mixtures contained 5 μMp-nitrophenyl acetate and 40 μg protein in 67 mM phosphate buffer (pH7.4). Reactions were performed at 37° C. The results of these assays areshown in FIG. 18.

The ability of recombinant RGD-containing HSAs to interact with αvβ3integrin was demonstrated. The results of this assay are shown in FIG.19. Lanes 1 and 4 each contained a mixture of 300 μg MDA-MD-435S (αvβ3positive) cell extract and 1.2 μg of RGD-rHSA-FN (rHSA containingfibronectin RGD sequence). Lanes 2 and 5 each contained a mixture of 300μg MDA-MD435S cell extract and 1.2 μg of RGD-rHSA-4C (rHSA containingRGD sequence held by tow pairs of disulfide bonds). Lanes 3 and 6 eachcontained mixture of 300 μg MDA-MD435S cell extract and 1.2 μg ofRGD-rHSA (wild type HSA). For the convenience of purification anddetection, all rHSAs were fused with c-terminal c-myc and His₆ tags. Forlane 1, 2, and 3, the αvβ3 complexes were immunoprecipitated withanti-αv antibody (CHEMICON, AB1930). rHSAs associated with αvβ3 weredetected by anti-c-myc antibody. The wild type rHSA was not detectable.For lane 4, 5, and 6, the poly-HIS-tagged rHSAs were pulled down withNi2+-charged chelating agarose. αvβ3 Associated with rHSAs was detectedby anti-αv antibody (CHEMICON, AB1930). Because the wild type rHSA doesnot interact with αvβ3 integrin, αv was not detectable at lane 6.

RGD-containing rHSAs were demonstrated to facilitate albumin-mediateduptake and/or binding of αvβ3 positive MDA-MB-435S cells. rHSAs werelabeled with NHS-Rhodamine (PIERCE 46102) at primary amines followingmanufacturer protocols. The products were thoroughly dialyzed anddesalted to remove free Rhodamine. The fluorescent dye labeled rHSAs (50pmol for rHSA, RGD-rHSA-FN and RGD-rHSA-4C) were added to culturedMDA-MB-435S cells (with 250 μl L-15 medium in 24-well plate). Cellularuptake and/or binding was monitored at 1, 2, 4, and 8 hours afterwashing with fresh un-labeled medium. Cellular uptake of albumin wasthen visualized by fluorescent microscopy. Cellular uptake and/orbinding of RGD-containing rHSAs could be clearly seen at the first hour.This process was difficult to observe for wild type rHSA at this point.However, albumin uptake for wild type rHSA could be observed after 4hours of incubation. Cellular uptake/binding of wt rHSA was stillsignificantly lower that that of RGD-containing rHSAs. TheRhodamine-labeled RGD-containing rHSA evenly distributed inside the cellwith centered bright dots. These results suggest that the endocytosiscontributes significantly to cellular uptake.

Albumin uptake and/or binding of rHSAs in HeLa cells was alsodemonstrated. The RGD sequence of RGD-rHSA-FN and RGD-rHSA-4C mildlystimulated albumin uptake and/or binding in HeLa cells. However, thiseffect was not as significant as the effects observed in MDA-MB-435Scells. These observations suggested a cell type-dependent function ofRGD-rHSAs. It has been reported that there is no αvβ3 type of integrinin HeLa cells. Nonetheless, HeLa cells are still able to bindfibronectin. Similar to the cellular uptake experiments for MDA-MB-435Scells, the fluorescent dye labeled rHSAs (50 pmol for rHSA, RGD-rHSA-FNand RGD-rHSA-4C) were added to cultured HeLa cells (with 250 μl EMEM in24-well plate). Cellular uptake and/or binding was monitored at 1, 4,and 8 hours after washing with fresh non-labeled medium. Cellular uptakeand/or binding of albumin was then visualized using fluorescentmicroscopy. Cellular uptake and/or binding of RGD-containing rHSAs andwt rHSA could be clearly observed at the first hour. This experimentsuggests that cellular uptake of albumin occurs for certain tumor cells.RGD binding and cellular uptake of albumin will both benefit thealbumin-mediated siRNA transfection in vitro and in vivo.

HSA was demonstrated to augment cellular uptake of fatty acid. BODIPYfatty acids (Invitrogen, D3823, 200 pmol) were incubated at 20° C. with1 μmol of defatted HSA or without HSA in 20 μl buffer with 20 mMTris-HCl (pH 6.8) and 150 mM NaCl for 15 minutes. The mixtures were thenadded to HeLa cells in 200 μl of EMEM. After washing the cells withfresh medium, cellular uptake and/or binding was visualized usingfluorescent microscopy after 12 hours of incubation. Cells incubatedwith defatted HSA exhibited more fatty acid uptake and/or binding thancells incubated without HSA. These results suggest that HSA can form acomplex with the fatty acid-conjugated fluorescent probe and facilitatedcellular uptake of the conjugate.

The ability of HSA to bind a fatty acid-conjugated peptide wasdemonstrated. A peptide designated faTraE (CHLDAHWKG; SEQ ID NO: 58) wasconjugated with fluorescein at the position of primary amine of Lysthrough MHS ester (synthesized by Sigma). This peptide (500 μmol) wasthen conjugated at the thiol group of Cys with 16-methanethiosulfonylhexadecanoic acid (Toronto Research Chemicals, Inc., 1:2 of molarration) in an acetone nitrile mixture to form HOOC—(CH₂)₁₅—S—S-peptide.The product was vacuum dried and resuspended in DMSO. The fattyacid-modified peptide (25 pmol) was incubated with defatted HSA (100pmol) in 10 μl buffer (Tris-HCl 20 mM pH6.8 and 150 mM NaCl) for 15minutes at room temperature. The reaction mixture was loaded to 0.5× TBEacrylamide gel and separated under 12 mA current. Thefluorescein-labeled DNAs were visualized under UV 352 nm. HSA complexedwith the fatty acid-conjugated faTraE peptide could be clearly observed.This experiment demonstrates the feasibility of using HSAs to deliverfatty acid-modified peptides.

The ability of fatty acid-conjugated peptides to transfect HeLa cells inthe presence of HSA was demonstrated. Fatty acid-conjugated faTraEpeptides and non-conjugated faTraE peptides (250 pmol) were added toseparate batches of HeLa cells (24-well plate with 250 μl of EMEMmedium). 250 Pmol of HSA was added to the incubation of some wells, butnot others. Cells incubated in the presence of HSA and the fatty-acidconjugated peptides were transfected. The transfection of unmodifiedpeptides to HeLa cells can not be observed under these conditions. Theseresults demonstrate the feasibility of transfecting cells using a fattyacid-conjugated transfection agent in the presence of HSA.

The ability of HSA to bind fatty acid-conjugated double strandedoligonucleotides was demonstrated. Thiol-modified 22 meroligonucleotides (CH₃—(CH₂)₅—S—S—(CH₂)₅-5′-DNA) (50 μM in 100 μl of H₂O)were reduced to HS—(CH₂)₅-5′-DNA with 10 mM DTT overnight at roomtemperature. The mixture was desalted to remove free DTT. Meanwhile,dithiodipyridine (10 μl of 10mM in acetonitrile) was mixed with16-mercaptohexadecanoic acids (10 μl of 10mM in DMSO) to modify the freethiol group of 16-mercaptohexadecanoic acids. The mixture was incubatedwith reduced DNA at room temperature for 60 minutes. The reaction wasthen desalted using a G-25 centrifugal column. The fatty acid-modifiedsingle-stranded oligonucleotides were annealed with 22 mer complementaryoligonucleotides labeled using the cy3 dye at their 5′ ends. The fattyacid-modified and cy3-labeled double stranded 22 mer oligonucleotides(25 pmol) were incubated with defatted HSA (30 pmol) in 10 μl buffer(Tris-HCl 20 mM pH6.8 and 150 mM NaCl) for 15 minutes at roomtemperature. The reaction was loaded to 0.5×TBE acrylamide gel andseparated under 12 mA. The cy3 labeled DNAs were visualized under UV 352nm. Fatty acid-conjugated oligonucleotides complexed with HSA could beclearly visualized based on their position on the gel. These resultssupport the feasibility of using HSAs to deliver fatty acid-modifiedoligonucleotides, such as siRNA. In other experiments, HSA was observednot to bind thiol-modified 22-mer oligonucleotides(CH₃—(CH₂)₅—S—S—(CH₂)₅-5′-DNA) or amine-modified 22-mer oligonucleotides(NH2-(CH₂)₁₂-5′-DNA). These results suggest that the carboxyl group ofthe fatty acid is critical for the binding of fatty acid-modified DNA.

The ability of fatty acid-conjugated oligonucleotides to transfect HeLacells in the presence of HSA was demonstrated. Fatty acid-conjugated andcy3 labeled oligonucleotides (synthesized as described above; 40 pmol)were added to HeLa cells (24-well plate with 250 μl of EMEM medium) inthe presence (80 pmol) and absence of HSA and the cells were incubatedin those mixtures. The modified oligonucleotides were able to transfectHeLa cells. Pre-incubating fatty acid-modified DNA with HSA mildlyincreased transfection efficiency, as assessed by the amount andbrightness of transfected cells in the assay system used.Cholesterol-modified oligonucleotides (40 pmol, ordered from IDT DNAInc.) were able to bind HSA as well as transfect HeLa cells. However,the transfection was toxic and caused cell death. This may be related tothe cellular membrane integration functions of cholesterol. In contrast,at the concentrations used here, the fatty acid-modifiedoligonucleotides did not exhibit cytotoxicity. As an negative control,amine-modified oligonucleotides (IDT DNA Inc.) with —(CH₂)₁₂-spacer wereused and exhibited only barley detectable transfected cells. The resultsof this experiment demonstrate the feasibility of using HSA-complexedfatty acid-conjugated oligonucleotides to transfect cells.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention can be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims include all such embodiments and equivalent variations.

What is claimed is:
 1. A recombinant animal albumin protein, proteinbeing a fusion protein having a ligand-binding protein domain insertedin place of a surface loop thereof.
 2. A recombinant human serum albuminprotein (rHSA) having a polypeptide inserted in place of a surface loopof native human serum albumin.
 3. The rHSA of claim 2, wherein thepolypeptide comprises a ligand-binding protein domain.
 4. The rHSA ofclaim 3, wherein the ligand is a ligand that occurs on the surface of acell of an animal.
 5. The rHSA of claim 3, wherein the ligand is aligand that occurs in a tissue of an animal.
 6. The rHSA of claim 3,wherein the domain is selected from the group consisting of anRGD-containing domain, an adrenomedulin domain, an endothelin-1 domain,a matrix metalloproteinase 9 binding peptide domain, a matrixmetalloproteinase 2 binding peptide domain, and an aminopeptidase Nbinding peptide domain.
 7. The rHSA of claim 3, wherein the domain hasan amino acid sequence selected from the group consisting of SEQ ID NOs:3-11.
 8. The rHSA of claim 2, wherein the surface loop is selected fromthe group consisting of (i) the loop defined by residues 53-62; (ii) theloop defined by residues 75-91; (iii) the loop defined by residues91-101; (iv) the loop defined by residues 168-177; (v) the loop definedby residues 245-253; (vi) the loop defined by residues 265-279; (vii)the loop defined by residues 278-289; (viii) the loop defined byresidues 360-369; (ix) the loop defined by residues 437-448; (x) theloop defined by residues 461-477; (xi) the loop defined by residues476-487; and (xii) the loop defined by residues 558-567 of SEQ ID NO: 1.9. A complex comprising the rHSA of claim 2 complexed with a compoundthat binds non-covalently therewith.
 10. The complex of claim 9, whereinthe compound is a conjugate of first molecule and a fatty acid.
 11. Thecomplex of claim 10, wherein the polypeptide comprises a ligand-bindingprotein domain.
 12. The complex of claim 11, wherein the ligand is aligand that occurs on the surface of a cell of an animal.
 13. Thecomplex of claim 11, wherein the ligand is a ligand that occurs in atissue of an animal.
 14. The complex of claim 11, wherein the domain isselected from the group consisting of an RGD-containing domain, anadrenomedulin domain, an endothelin-1 domain, a matrix metalloproteinase9 binding peptide domain, a matrix metalloproteinase 2 binding peptidedomain, and an aminopeptidase N binding peptide domain.
 15. The complexof claim 11, wherein the domain has an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 3-11.
 16. The complex of claim 10,wherein the surface loop is selected from the group consisting of (i)the loop defined by residues 53-62; (ii) the loop defined by residues75-91; (iii) the loop defined by residues 91-101; (iv) the loop definedby residues 168-177; (v) the loop defined by residues 245-253; (vi) theloop defined by residues 265-279; (vii) the loop defined by residues278-289; (viii) the loop defined by residues 360-369; (ix) the loopdefined by residues 437-448; (x) the loop defined by residues 461-477;(xi) the loop defined by residues 476-487; and (xii) the loop defined byresidues 558-567 of SEQ ID NO:
 1. 17. The complex of claim 10, whereinthe first molecule is selected from the group consisting of apolynucleotide, a polypeptide, and a drug other than a polynucleotide ora polypeptide.
 18. The complex of claim 10, wherein the first moleculeis selected from the group consisting of a radiolabeled compound and animaging agent other than a radiolabeled compound.
 19. The complex ofclaim 10, wherein the fatty acid is a C₁₆-C₂₀ fatty acid.
 20. Acomposition for delivering a first molecule to a cell of a human, thecomposition comprising a recombinant human serum albumin (rHSA)non-covalently complexed with a conjugate, wherein the conjugatecomprises a fatty acid moiety conjugated with the first molecule. 21.The composition of claim 20, wherein the composition containssubstantially no first molecules not complexed with the recombinanthuman serum albumin (rHSA).
 22. The composition of claim 20, wherein therecombinant human serum albumin (rHSA) is non-covalently complexed witha second conjugate, wherein the second conjugate comprises a secondfatty acid moiety conjugated with a compound that binds specificallywith a target molecule that occurs on the surface of the cell.
 23. Thecomposition of claim 20, wherein the recombinant human serum albumin(rHSA) is non-covalently complexed with a second conjugate, wherein thesecond conjugate comprises a second fatty acid moiety conjugated with acompound that binds specifically with a target molecule that occurs in atissue that includes the cell.
 24. The composition of claim 20, whereinthe human albumin is a rHSA, the rHSA being a fusion protein having apolypeptide inserted in place of a surface loop of human serum albumin.25. The composition of claim 24, wherein the polypeptide comprises aligand-binding protein domain.
 26. The composition of claim 25, whereinthe ligand is a ligand that occurs on the surface of a cell of ananimal.
 27. The composition of claim 25, wherein the ligand is a ligandthat occurs in a tissue of an animal.
 28. The composition of claim 25,wherein the domain is selected from the group consisting of anRGD-containing domain, an adrenomedulin domain, an endothelin-1 domain,a matrix metalloproteinase 9 binding peptide domain, a matrixmetalloproteinase 2 binding peptide domain, and an aminopeptidase Nbinding peptide domain.
 29. The composition of claim 25, wherein thedomain has an amino acid sequence selected from the group consisting ofSEQ ID NOs: 3-11.
 30. The composition of claim 24, wherein the surfaceloop is selected from the group consisting of (i) the loop defined byresidues 53-62; (ii) the loop defined by residues 75-91; (iii) the loopdefined by residues 91-101; (iv) the loop defined by residues 168-177;(v) the loop defined by residues 245-253; (vi) the loop defined byresidues 265-279; (vii) the loop defined by residues 278-289; (viii) theloop defined by residues 360-369; (ix) the loop defined by residues437-448; (x) the loop defined by residues 461-477; (xi) the loop definedby residues 476-487; and (xii) the loop defined by residues 558-567 ofSEQ ID NO:
 1. 31. The composition of claim 20, wherein the firstmolecule is selected from the group consisting of a polynucleotide, apolypeptide, and a drug other than a polynucleotide or a polypeptide.32. The composition of claim 20, wherein the first molecule is selectedfrom the group consisting of a radiolabeled compound and an imagingagent other than a radiolabeled compound.
 33. The composition of claim20, wherein the fatty acid is a C₁₆-C₂₀ fatty acid.